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Cancer preventive Effect of Morinda citrifolia (Noni) fruit juice against

the AflatoxinB1-induced genotoxicity in human peripheral lymphocytes
in vitro.
MD. SULTAN AHMAD, SHEEBA, AFSAR ALI AND KANCHAN B. RAI.
Department of Zoology, S.N. (P.G.). College, Azamgarh, U.P.276001

ABSTRACT
Morinda citrifolia (Noni) has been used in folk medicine by Polynesians for over 2,000 years and is reported to have a broad
range of therapeutic effects, including anticancer activity. The exact mechanism of action is unknown. The hypothesis is
generated from the experiment that Morinda citrifolia possesses a cancer preventive effect at the initiative stage of
carcinogenesis. The antigenotoxic potential of Noni juice (NJ) was demonstrated on the aflatoxin B1 induced genotoxicity. In
vitro studies were carried on human lymphocyte culture. We have used chromosomal aberration (CA), sister chromatid
exchange (SCE) and cell cycle kinetics (CCK) with and without S9 mix. as markers in this experiments. Four doses viz., 200,
250, 300, 350 l/ml per culture were selected and found that NJ significantly reduces the frequencies of chromosomal
aberration, sister chromatid exchanges and enhances RI in vitro .It was also noticed that the antigenotoxic potential of NJ
shows dose response relationship. The results suggest that NJ was a potent anticarcinogen may contribute to the cancer
prevention.

Key words:

Morinda citrifolia (Noni), Noni Fruit juice (NJ), chromosomal aberration, Sister Chromatid Exchange,

anticarcinogen.

INTRODUCTION:
Noni is the common name for Morinda citrifolia. It is a medicinal plant called Indian mulberry in India, bajitian in
China, Nono in Tahiti, and noni in Hawaii (Morton J, 1992.). In India it is distributed throughout Tamil Nadu and Kerala in
South India, especially coastal region and also in the Mangalore area of Karnataka. Among the medicinal plants discovered
by the ancestors of Polynesians, Morinda citrifolia L(Noni) is one of the traditional folk medicinal plants that has been used
for over 2000 years in Polynesia. It has been reported to have a broad range of therapeutic effects like anti-cancerous, antitumor, and have nutritional value. Noni has an abundance of micronutrients. It has also been used by the native Tahitians for
over 2000 years as a nutritional supplement to treat diseases and promote general good health (Abbott & Shimazu, 1985;
Degener, O. 1973).
Noni plants have 160 identified chemicals, the major components are terpene, scopoletin, octoanoic acid, potassium,
vitamin C, terpenoids, alkaloids anthraquinones (such as nordamnacanthal, morindone, rubiadin, and rubiadin--methyl
ether anthraquinone glycosides), sitosterol, carotene, vitamin A, flavone glycosides, linoleic acid, alizarin, amino acids,
cacubin, L-asperuloside, caproic acid, caprylicacid, ursolic acid, rutin, and a putative proxeronine polysaccharides, and
alkaloids. Dr Neil Solomon summarized 15,000 cases of NJ users, and found the total effective rate of NJ on various health
problems, including cancer to be 78% effective. NJ may possess a cancer preventive effect at the initiation stage of
carcinogen and /or antioxidant activity.
The anticancer activity from alcohol-precipitate of Noni fruit juice (Noni-ppt on to lung cancer in c57 B1/6 mice has
been presented in the 83 Annual Meeting of American Association for Cancer Research. The NJ significantly increased the
life of mice up to 75% with implanted Lewis lung carcinoma as compared with the control mice (Hirazumi et al., 1994). It
was concluded that the NJ seems to suppress tumor growth directly by stimulating the immune system (Hirazumi et al.,
1996). Improved survival and curative effects occurred when Noni-ppt was combined with sub optimal doses of the standard
chemotherapeutic agents such as adriamycin (Adria), cisplatin(CDDP), 5-flourouracil (5-FU) and vincristine (VCR),
suggesting important clinical application of Noni-ppt as a supplemental agent in cancer treatment (Hirazumi and Furusawa,
1999). These results indicated that the Noni-ppt might enhance the therapeutic effect of anticancer drugs. Therefore, it may
be a benefit to cancer patients by enabling them to use lower doses of anticancer drugs to achieve the same or even better
results. Wang et al. (2002) demonstrated that the cytotoxic effect of Tahitian Noni Juice (TNJ) on cultured leukemia cell line
at various concentrations. They also observed the synergistic effects of TNJ with known anticancer drugs. At a sub-optimal
dose, both prednisolone and TNJ could induce apoptosis. When the dose of prednisolone was fixed, the dose of TNJ
increased. Therefore TNJ is able to enhance the efficacy of anticancer drugs such as predinosolone. When a single dose of
taxol induced a lower percentage of apoptosis in leukemia cells, TNJ enhanced the rate of apoptosis. Hiramatsu et al. (1993)
reported effects of over 500 extracts from tropical plants on the K-Ras-NRK cells. The Ras oncogene is believed to be
associated with the signal transduction in several human cancers such as lung, colon, pancreas, and leukemia. Two glycosides
extracted from Noni-ppt were effective in inhibiting cell transformation induced by TPA or EGF in the mouse epidermal JB6
cell line. The inhibition was found to be associated with the inhibitory effects of these compounds on AP1 activity. The

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compounds also blocked the phosphorylation of c-Jun, a substrate of JNKs, suggesting that JNKs are the critical target for the
compounds in mediating AP1 activity and cell information (Liu et al., 2001).

MATERIALS AND METHODS:

Experiment was performed using the technique of Moorehead et.al (1960), for metaphase chromosome analysis and
for detection of chromosomal aberration analysis (CAs). Human lymphocyte cultures were set by adding 0.5 ml of whole
blood (from two adult and healthy donors, occupationally not exposed to mutagens) to 4.5 ml of RPMI 1640 (Gibco, USA),
antibiotics (Penicillin and streptomycin 100 IU/ml each; Hoechst) and L. Glutamine (1 mM; Gibco, USA). Lymphocytes
were stimulated to divide by adding 0.1 ml of phytohaemagglutinin M (PHA M, Gibco). The cultures were incubated at
37oC with 5% CO2 for 72 hours in dark. Aflatoxin B1 at a final concentration of 50 g was added at 0 hour and kept for 24,
48 and 72 hours of duration, which served as positive control. Subsequently, desired test chemical were added along with
Aflatoxin B1 and the cultures were kept for 24, 48 and 72 hours. Noni juice and Aflatoxin B1 were prepared in DMSO. In the
metabolic activation experiments cultures were treated with S9 mix (0.8 ml.), the S9 mix was freshly prepared as per the
standard procedures of Maron and Ames (1983). The S9 fraction was complemented by the addition of 5 M NADP and 10
M glucose 6- phosphate just before use. Colchicines (0.20 g/ml, Micro lab) were added to the cultures 2.5 hours prior to
harvesting. The cells were collected by centrifugation (10 min, 1200 rpm), hypotonic treatment (0.075M KCl) was given for
10-12 min at 370C and the recollected cells after centrifugation were fixed in methanol: acetic acid (3:1). DMSO and
Aflatoxin B1 were uses as negative and positive controls, respectively. Preparation of slides, staining and scanning was
done under code. A total of 300 well - spread metaphases were analyzed per treatment per duration for all types of chromatid
and chromosome type of aberrations. Aberrations were scored as per Hundal, et al ( 1997). Analysis of SCE was carried out
following the fluorescent plus Giemsa technique of Perry and Wolfs (1974). The cells in the cultures were exposes to 5bromo-2-de oxyuridine (BrdU 2 g/ml; Sigma) after 24 hours of initiation of culture. The test compounds with same
concentrations as in the case of CA analysis were added together with the BrdU. To minimize photolysis of BrdU another 48
hours cultures were maintained in the dark. After 90 min. of this pulse treatment the cells were spun down and the
supernatant discarded. The cells were washed twice to remove any traces of the drug, phytoproducts and the liver
metabolites. Finally the cell pellets were re-suspended in fresh medium supplemented with fetal calf serum, antibiotics and
BrdU, and kept for another 24 hours in the dark at 37oC.
One day old slides were stained in Hoechst 33258 stain (Sigma 0.5 g/ml), exposed to UV lamp (254 nm) for 30
min. and incubated in 2X SSC (0.3 M NaCl, 0.03M Sodium citrate; pH 7.0) at 60 oC for 90 min and stain for sister chromatid.
The slides were coded prior to scoring and 50 well- spread metaphase cells were scanned per concentration and the number
of exchanges scored (Hundal, et al., 1997). Cells undergoing 1st (M1), 2nd (M2) and 3rd (M3) metaphase divisions were
detected with BrdU Harlequin technique for differential staining of metaphase chromosome by studying 200 metaphases
for each combination and duration. The replication index (RI), an indirect measure of studying cell cycle progression, was
calculated by applying the following formula (Tice, et al., 1976).
RI =

M1x1+M2x2+M3 x3
100

RESULT AND DISCUSSION:

Noni is a medicinal plant that has antioxidant activity, may protect individuals from free oxygen radicals and consequent
lipid peroxidation. In this experiment we have notices that Aflatoxin B1 treatment caused aberrations from 30.50 to 60.0% as
the durations increases, when NJ are used it reduces from 19.75 to 50.0% at lower concentration and 19.0 to 46.5% at the
highest concentration (Table-1).When culture were augmented with S9 mix, effects of NJ has shown more effective in
reducing the percent of aberration. The range and mean values of SCE get reduced with NJ in the absence as well in the
presence of metabolic activation (Table-2). The replication indices, which are reduced due to Aflatoxin B1 treatment level
(1.44), is elevated to 1.58, thus bringing the cell proliferation nearly back to near about normal of 1.60. The S9 activation has
follow the above trend (Table-3 ).
In general, consuming fruits and vegetables reduces free radicals induced oxidative damage and the consequent lipid
peroxidation and therefore reduce the cancer risk (Wang and Leiher, 1995; Diplock et al., 1998). It is believed that fruits and
vegetables are major sources for antioxidants (Weisburer et al., 1997; Nishikimi et al., 1972). Noni is a medicinal plant that
helps the human in different health conditions. It was believed that the Noni fruit juice contained significant level of
antioxidants. This has been proved scientifically by the analysis of NJ. The study was designed to measure how the NJ
scavenged superoxide anion radicals (SAR) and quenched lipid peroxides (LPO) by TNB assay and LMB assay, respectively
(Auerbach et al., 1992; Wang and Su, 2001). SAR scavenging activity was examined in vitro by Tetrazolium nitroblue
(TNB) assay. The SAR scavenging activity of NJ was compared to that of three known antioxidants; vitamins C, grape seed
powder, and pyncogenol at the daily dose per serving level recommended by US RDAs or manufacturers recommendations.
Under the experimental conditions the SAR scavenging activity of NJ was shown to be 2.8 times that of vitamin C, 1.4 times

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that of pyncogenol and1.1 times that of grape seed powder. Therefore NJ has a great potential to scavenge reactive oxygen
free radicals (Wang and Su, 2001).
The major ingredient in NJ and fruit has been safely consumed in other parts of the world for several hundred years.
The interactions of carcinogens, free oxygen radicals, and LPO may be changed by NJ. The mechanism of the cancer
preventive effect of NJ needs further study.
In conclusion, on the basis of our preliminary data, NJ may possess a cancer preventive effect at the initiation stage
of chemical carcinogenesis. It serves as a good liquid nutritional supplement. NJ may help to prevent cancer and other
diseases, and maintaining overall good health.

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Percent Aberration
Metaphase

Total

300
200

30.00
36.25

28.00
32.50

25.50
30.20

12.50
20.30

38.00
50.50

0.38 0.02
0.50 0.02

Af B1+ NJ0

-S9
+S9

300
200

25.35
32.50

23.22
30.35

20.10
25.00

15.20
23.50

35.30
48.50

0. 35 0.02
0 .48 0.02

Af B1+ NJ1

-S9
+S9

300
200

22.30
28.00

20.19
25.35

20.50
24.50

12.30
21.20

32.80
45.70

0.32 0.02
0.45 0.02

Af B1+ NJ2

-S9
+S9

300
200

20.50
27.32

19.25
23.30

18.30
23.70

12.20
18.50

30.50
42.20

0.30 0.02
0.42 0.02

Af B1+ NJ3

-S9
+S9

300
200

18.50
25.50

17.24
21.35

17.30
22.50

10.20
18.50

27.50
41.00

0.27 0.02
0.41 0.02

-S9
+S9

300
200

2.50
3.00

2.00
2.50

2.50
2.25

0.75
1.00

3.25
3.25

0.03 0.02
0.03 0.02

Normal

-S9
+S9

300
200

3.50
4.20

2.00
3.70

2.75
3.00

0.35
0.50

3.10
3.50

0.03 0.02
0.03 0.02

DMSO(5 g/ml)

-S9
+S9

300
200

4.00
4.50

1.75
2.00

3.50
3.00

0.75
1.00

4.25
4.00

0.04 0.02
0.04 0.02

Excluding
Gap

-S9
+S9s

Treatment

Metabolic
Activation

Chromosome

Types of aberration(%)
Chromatid

Including Gap

Metaphase scanned

Table-1. Analysis of C.A. after treatment of Aflatoxin B1 and NONI JUICE (NJ )in absence as well as presence of S9
mix in vitro.

Aberration/Cell
SE

AflatoxinB1(50g/ml)

Aflatoxin B1 + NJ

Control

Normal + F2

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Aflatoxin B1(50 g/ml)

48

Total SCE

Range

SCE/Cell SE

Metaphase
scanned

Treatment

Metabolic
activation

Duration (h)

Table-2. Analysis of SCE, after treatment of Aflatoxin B1 and Noni juice (NJ) in vitro with and without metabolic
activation.

-S9
+S9

50
50

175
210

1-10
1- 9

3.50 0.50
4.20 0.80

-S9
+S9

50
50

152
170

1-10
2-12

3.04 0.50
3.40 0.70

-S9
+S9

50
50

135
165

0-9
1-9

2.70 0.40
3.30 0.50

-S9
+S9

50
50

121
141

0-7
0-9

2.42 0.30
2.82 0.40

-S9
+S9

50
50

110
130

0- 6
0- 9

2.20 0.20
2.60 0.40

-S9
+S9
-S9
+S9
-S9
+S9

50
50
50
50
50
50

57
70
65
85
70
95

0-6
0-6
0-5
0-7
0-5
0-6

1.14 0.14
1.40 0.14
1.30 0.12
1.70 0.20
1.40 0.14
1.90 0.20

Aflatoxin B1 + NJ
Af B1+ NJ0
48
48
Af B1+ NJ1

48
Af B1+ NJ2
48
Af B1+ NJ3
Control

Normal + F2

48

Normal

48

DMSO(5 g/ml)

48

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Table-3. Analysis of Cell cycle kinetic after treatment of Aflatoxin B1 and Noni juice(NJ) in vitro with and without
metabolic activation.
Percent Aberration Metaphase

Replication
Index (RI)

2*3 Chi square test

Treatment
Cell scored
200
200

Metabolic
activation
-S9
+S9

Af B1+ NJ0

200
200

Af B1+ NJ1

200
200

Af B1+ NJ2

200
200

Af B1+ NJ3

200
200

Aflatoxin B1(50
g/ml)
Aflatoxin
Flavonoids

B1

M1

M2

M3

54
57

39
35

04
05

1.44
1.42

Significant
Significant

-S9
+S9

51
53

38
36

08
07

1.51
1.46

Signficant
Signficant

-S9
+S9

52
54

37
38

09
12

1.53
1.66

Signficant
Signficant

-S9
+S9

48
50

40
42

13
08

1.67
1.58

Not Signficant
Signficant

-S9
+S9

49
51

36
40

15
09

1.66
1.58

Signficant
Signficant

40
41
42

35
37
36

18
15
14

1.64
1.60
1.56

Not Signficant
normal

Control
Normal + NJ
Normal
DMSO(5 g/ml)

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200
200

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